Ab Initio Modeling and Experimental Assessment of Janus Kinase 2 (JAK2) Kinase-Pseudokinase Complex Structure

Xiaobo Wan, Yue Ma, Christopher L McClendon, Lily Jun-shen Huang and Niu Huang

PLOS Computational Biology, 2013, vol. 9, issue 4, 1-13

Abstract: The Janus Kinase 2 (JAK2) plays essential roles in transmitting signals from multiple cytokine receptors, and constitutive activation of JAK2 results in hematopoietic disorders and oncogenesis. JAK2 kinase activity is negatively regulated by its pseudokinase domain (JH2), where the gain-of-function mutation V617F that causes myeloproliferative neoplasms resides. In the absence of a crystal structure of full-length JAK2, how JH2 inhibits the kinase domain (JH1), and how V617F hyperactivates JAK2 remain elusive. We modeled the JAK2 JH1–JH2 complex structure using a novel informatics-guided protein-protein docking strategy. A detailed JAK2 JH2-mediated auto-inhibition mechanism is proposed, where JH2 traps the activation loop of JH1 in an inactive conformation and blocks the movement of kinase αC helix through critical hydrophobic contacts and extensive electrostatic interactions. These stabilizing interactions are less favorable in JAK2-V617F. Notably, several predicted binding interfacial residues in JH2 were confirmed to hyperactivate JAK2 kinase activity in site-directed mutagenesis and BaF3/EpoR cell transformation studies. Although there may exist other JH2-mediated mechanisms to control JH1, our JH1–JH2 structural model represents a verifiable working hypothesis for further experimental studies to elucidate the role of JH2 in regulating JAK2 in both normal and pathological settings. Author Summary: Protein-protein interactions (PPIs) are essential to cellular signal transduction, and structural information about PPIs is crucial for understanding of how cellular machinery functions at the atomistic level. However, both experimental structural determination and computational prediction of PPI are challenging. In the cytoplasmic tyrosine kinase JAK2, a pseudokinase domain (JH2) negatively regulates kinase activity of its adjacent catalytic kinase domain (JH1). A gain-of-function mutation within JH2 is found in the majority of patients with myeloproliferative neoplasms, and is sufficient to cause similar diseases in murine models. Here we combined an informatics-guided protein-protein docking method with molecular dynamics simulation to construct and refine the JAK2 JH1–JH2 complex, and validated our model with mutational studies. Our modeled structure suggests that JH2 auto-inhibits JAK2 kinase activities by blocking the movements of the activation loop and the αC helix of JH1, but awaits further validation by a detailed structure of the full-length JAK2 protein.

Date: 2013
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Persistent link: https://EconPapers.repec.org/RePEc:plo:pcbi00:1003022

DOI: 10.1371/journal.pcbi.1003022

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